1. Field of the Invention
The invention relates to analytical procedures for investigating analyte species in a fluid sample. More specifically, the invention relates to methods and apparatus for separation and manipulation of analytes, and their application in diagnostic qualitative and quantitative procedures.
2. Brief Description of Related Art
Electrormigration separation processes, such as capillary zone electrophoresis and micellular electrokinetic capillary chromatography and other similar processes, are known and are widely used. For example, these processes are used in research and in various testing applications such as separations of proteins and DNA fragments. Control, and particularly fine control, of these processes is an-area of ongoing research and development efforts, as manipulation of quantities of analyte species for separation and qualitative and quantitative analysis is recognized as a significant technical challenge, but one potentially yielding great benefits in scientific endeavors and applications in areas such as healthcare that could potentially greatly benefit humankind.
Taking as an example capillary electrophoresis (CE), and with reference to FIG. 1 which is a generalized schematic diagram of CE, a capillary tube 12 having a longitudinal axis is disposed between two fluid wells positioned at its ends. An electrolyte solution, such as a buffer solution, is contained in the wells and an interior channel defined by the capillary. A cathode 14 is positioned in fluid communication with the electrolyte solution at one end and the anode 16 at the other. An applied voltage potential across the cathode and anode gives the potential or voltage profile 20 shown in the diagram. This is a straight line having a slope equal to the magnitude of the potential difference over the length of the channel. The electric field 22 created in the capillary is a constant throughout its length as shown. This of course is because the channel is of uniform cross-section and the voltage drop per unit of distance along the longitudinal axis of the channel is a constant value. In other words, the derivative of the function defined by the voltage as a function of position along the axis is a constant and, accordingly, the field generated is also of a constant intensity along the longitudinal axis.
Moreover, as is known in electrophoretic processes, an electrolyte solution, which provides a medium in which analytes to be investigated, e.g. separated and identified, are resident during separation, can be subject to electroosmotic flow, and such flow in small channels is a plug or bulk flow. Further, it has been recognized that electroosmotic bulk flow of electrolyte fluid in electrophoretic processes can be used to enhance separation, and also to do other things, such as to move analytes around within a electrophoretic separation apparatus. For example, U.S. Pat. No. 5,151,164, U.S. Pat. No. 5,180,475 and U.S. Pat. No. 5,320,730 disclose methods and apparatus for controlling the electroosmotic bulk flow of electrolyte solutions in electrophoretic processes, and the disclosures of these patents are hereby incorporated herein by reference.
It is known that control of the polarity and magnitude of charge accumulation adjacent to the inner surface of containments (such as fused silica capillary tubes) wherein the electrolyte solution dwells during electrophoretic separations (known in the art as the zeta potential) effectively controls electroosmotic bulk flow in small channels. This has been explained in terms of viscous coupling of molecules in solution with accumulations of charged molecules adjacent to the inner surface, such accumulated charged molecules being actuated in a longitudinal direction toward a cathode or anode (depending on polarity) by the electric field within the containment. As mentioned, this effect has been found to create a reproducible xe2x80x9cplugxe2x80x9d flow of electrolyte solution, and that this flow is relatively stable. Thus, in electrophoretic separation processes, analytes may be carried along in the bulk flow of the electrolyte solution, which fact can be used to move analytes, and assist in the separation of distinct analyte species based on differing mobilities of constituent molecules in the electrolyte solution (and any other media, such as a gel, which also may be present) within the separation channel.
For example, it is known that resolution of discrete analyte species having similar mobilities is enhanced by balancing electroosmotic flow against electrophoretic migration. Depending on whether cations or anions are of interest, the polarity and magnitude of the zeta potential is selected by applying an appropriate potential at the outer surface of a capillary having appropriate dielectric properties. Applying sufficient external potential of the same polarity as the molecules collecting at the surface overcomes the electroosmotic flow regime spontaneously occurring in the direction of electrophoretic migration by reversing the polarity of the inner surface, causing the spontaneously accumulating molecules to disperse and those of opposite polarity to migrate to the inner surface, setting up bulk flow in the opposite direction. As will be recognized, by application of appropriate potential external to the capillary, the electroosmotic flow within it can be increased, decreased, stopped and reversed.
However, manipulation of the electromagnetic forces effecting electrophoretic migration of charged species, which gives rise to separation, is less well recognized and understood as a tool in enhancing separation. Some work in this area has been done. For example, two published articles discuss localized modification of the intensity of the electric field for the purpose of improving resolution of analyte species. In an article [Kroegler and Ivory] published in [Journal of Chromatography A, 229 (1996) 229-236] there is a disclosure of a separation system wherein an electric field intensity which varies as a function of position, providing a sloping intensity profile along a longitudinal axis of the apparatus, is balanced against a pump-induced flow providing a counter-acting force. However, the system disclosed requires two flow fields, separated by a length of dialysis membrane tubing. One field comprises an electrolyte solution in which the electric field is propagated and is bounded by a trumpet shaped containment. The flared shape of the containment provides a cross section which varies as a non-linear continuous function of position along a longitudinal axis, and therefore gives an electric field within the channel that varies in intensity as a continuous function of position. Ions can cross the membrane, and the field extends within the other flow regime, but analyte molecules cannot pass out of the membrane into the electrolyte solution outside it. A buffer solution containing the analyte sample to be separated is pumped through the tubing in a direction opposite to the electrophoretic migration of the molecules of interest, and differences in electrophoretic mobility causes analyte species to xe2x80x9cfocusxe2x80x9d at differing equilibrium points where the electrophoretic force balances the bulk flow force for each molecule of a species. In other words the molecules will stop at different points corresponding to balanced forces acting on them, and like molecules will stop at like points along the longitudinal axis.
In the other published reference [Huang and Ivory] [Anal. Chem. 1999, 71, 1628-1632], the electric field intensity is altered locally by 50 electrodes spaced along the longitudinal axis of the apparatus. A potential is applied at each electrode, the result taught being to provide a gradient in intensity. Like the previous article, the analyte of interest is contained in a flow regime separated from a second flow regime by dialysis tubing. The tubing is packed with gel which provides an opposition to flow of fluids and to electrophoretic migration though the tubing. A bulk flow in opposition to electrophoretic migration is taught, with a focusing effect similar to that discussed above. However, the electric field intensity appears to:be a stair-step function of position (49 equilibrium positions) with discontinuities where the width of the electrodes create small zones of constant electric potential. As can be seen in FIG. 2, which is a schematic representation of at least a portion of the apparatus described in the reference, the potential (voltage) profile 20 is a construct of segments having differing slopes depending upon the voltages applied at each electrode 24 of the 50 electrode array. The electric field intensity profile 22 appears to be a distribution of xe2x80x9cplateausxe2x80x9d forming the tread portion of the stair step function, the height of each correlating with the slope of the voltage profile at the same location along the longitudinal axis 26 of the channel 28. The intensity (magnitude) of the electric field being constant between electrodes, there is no counter, or xe2x80x9crestoringxe2x80x9d force regime between electrodes tending to focus differing analyte species of slightly differing mobilities to differing locations on the xe2x80x9cplateauxe2x80x9d or stair step of constant field intensity over the space between adjacent electrodes. Therefore, it appears that unless the potential differences between electrodes are sufficiently small, individual analyte species cannot be separated. If numerous analyte species are present in the sample and the differences in mobilities are both small and large, multiple runs will be required.
At least some difficulties appear to be inherent in the two approaches disclosed in the journal references mentioned above, the first being a need to provide a separate flowpath for the analytes and buffer, as distinguished from other fluid media within a separation apparatus (in both cases separated by a membrane tubing), which other fluid media acts as a coolant in addition to propagating the electric field. Another is the stair step function of electric field intensity mentioned does not allow separation of more analyte species than the number of electrodes provided; and two or more of similar mobility could be indistinguishably mixed at the location of a single stair xe2x80x9cstep.xe2x80x9d
It has been recognized that the effectiveness of separation and the isolation of individual analyte species for identification can be enhanced by enabling more precise control over separation processes. The addition of other tools for manipulation of analyte species to a separation system has also been recognized as desirable to enable an electrophoretic separation system to perform tasks previously not undertaken in detection and analysis of disease states, and other analytical endeavors. One attribute of a separation system recognized as very desirable is to separate a sample containing hundreds if not thousands of analyte species, many of which have mobilities which are very similar, in a single separation run in a single channel.
The system of the invention accordingly provides an electromobility focusing system, which in a broad sense is defined herein as a system for separation and/or concentration of individual analyte species according to their electrophoretic mobilities in a medium, and in a more detailed aspect comprising a liquid-phase controlled channel electrophoresis separation system configured to separate at least one discrete analyte species from an analyte sample comprising:
1) a separation channel defined by a confinement enclosing an interior channel volume, said separation channel having first and second ends and a longitudinal axis, and said separation channel-being configured to contain an electrolyte solution within the interior channel volume, wherein the separation channel provides the only flowpath for both the analyte sample and the electrolyte solution;
2) a continuous electric field intensity gradient generator configured to apply a electric field intensity gradient within the separation channel along the longitudinal axis over at least a portion of the separation channel intermediate the first and second ends, the intensity of electric field generated varying as a continuous function of location along the longitudinal-axis, whereby electrophoretic migration of an analyte species within the separation channel is actuated by a force that varies with position along the longitudinal axis as a continuous function of position along the longitudinal axis within said portion of the separation channel;
3) an electroosmotic flow generator configured to generate an electroosmotic flow along thee longitudinal axis of the separation channel, which electroosmotic flow is variable as to at least one of: (i) the magnitude of the flow, and (ii) the direction of the flow, to enhance separation of said at least one analyte species by enabling control of an interaction of forces acting on it created by the continuous electric field tensity gradient generator and the electroosmotic flow generator.
In a more detailed aspect, such a system can be provided wherein the electroosmotic flow generator comprises a power supply and a distributed source of potential positioned adjacent said containment on an exterior surface. As a result, the zeta potential of an interior surface in fluid contact with the separation channel can be altered by at least one of: a) applying a potential, and b) altering at least one of: (i) the magnitude, and (ii) polarity, of potential applied, to the distributed source of potential from the power supply.
In a further more detailed aspect, the continuous electric field intensity gradient generator can further comprise: 1) a cathode positioned adjacent one of the first and second ends of the separation channel; 2) an anode positioned adjacent the other of the first and second ends of the separation channel; 3) a power supply in electric communication with the cathode and the anode; 4) a continuously varying resistor, which can be a contour resistor in fluid communication with the separation channel along at least a portion of the longitudinal axis intermediate the first and second ends, said resistor having a resistance that varies as a continuous function of position along the longitudinal axis of the separation channel, whereby an electric potential in the electrolyte fluid varies a non-linear continuous function of position along the longitudinal axis of the separation channel, and as a result the electric field intensity varies as a continuous functions of position along the longitudinal axis over at least a portion of the separation channel intermediate the first and second ends.
A continuously varying resistor in fluid communication with the separation channel along at least a portion of the longitudinal axis intermediate the first and second ends comprises a resistor having a resistance that varies as a continuous function of position along the longitudinal axis of the separation channel, whereby an electric potential in the electrolyte fluid varies as a non-linear continuous function of position along the longitudinal axis of the separation channel, and as a result the electric field intensity varies as a continuous function of position along the longitudinal axis over at least a portion of the separation channel intermediate the first and second ends. Such a resistor can comprise a contour resistor which contacts the fluid within the channel by forming a part of the channel wall, or the continuously varying resistor can comprise a filament within the separation channel, or the continuously varying resistor can comprise some other variable, such as a packing within the separation channel that varies in resistivity as a continuous function of position along the longitudinal axis. In further detail, a contour resistor can comprise a conductive material having a cross sectional shape which varies as a continuous function of position along the longitudinal axis. Alternatively, the contour resistor can be configured so that it has a material property that varies as a continuous function of position along said longitudinal axis.
In another more detailed aspect, a fluid electrolyte solution can be disposed in the separation channel, and the electrolyte solution can comprise a buffer solution. The system can further comprise a micellular dispersion or gel disposed in the separation channel. Alternatively the system can comprise a polymeric solution disposed in the separation channel.
In further detail, the containment can be configured to provide a high aspect, substantially rectangular cross-sectional shape for the separation channel. The electroosmotic flow generator can comprise a first plate disposed adjacent one side of the containment and be configured to alter the zeta potential on an interior surface of the separation channel adjacent the first side of the containment, and a second plate adjacent a second side of the containment configured to alter the zeta potential on an interior surface of the containment adjacent the second side of the containment. Said first and second plates can comprise distributed resistors laid down by a screening mask, or other deposition technique.
In another detailed aspect, the system can further comprise a first orientation electric field generator. The first orientation electric field generator can comprise an electroosmotic flow generator as set forth above, wherein the first plate and the second plate are brought to different potentials so as to create a transverse or alignment electric field configured to align bipolar molecules in directions normal to the first and second plates. The orientation electric field can be made to oscillate at a selected frequency. The system can further comprise a second orientation electric field generator configured for generating a second orientation electric field acting in a direction normal to the first orientation electric field, wherein the first and second orientation electric fields can be varied to orient bipolar molecules to a selected orientation by cooperation between the first and second orientation alignment electric fields.
In a further detailed aspects, the system can further comprise: i) a detector configured for detecting analyte species in said separation channel, said detector being positioned intermediate the first and second ends of said separation channel; ii) a steering valve in fluid communication with the separation channel, said steering valve comprising a connecting channel and configured to selectively divert fluid containing analyte species from said separation channel at a location intermediate the first and second ends of the separation channel into the connecting channel, and a second separation channel adapted for containing electrolyte fluid and analyte species, said second separation channel having a longitudinal axis and a first end and a second end, said second separation channel being in fluid communication with the connecting channel of said steering valve at a location intermediate said first and second ends, said second separation channel further comprising a second electric field generator configured for moving analyte species along the second separation channel by at least one of electrophoretic migration and electroosmotic flow; ii) an analyte concentrator located in said second-separation channel intermediate the first and second ends.
In a further detailed aspect the analyte concentrator can comprise a line source of electropotential and an isolated ground, whereby an electric field generated by the second electric field generator can be locally altered so as to focus an analyte species at a location intermediate the first and second ends of the second separation channel. The analyte concentrator can further comprise a first electrode positioned at a first point along the longitudinal axis of the second separation channel, said first electrode being connected to said isolated ground, and a second electrode positioned at a second point along said longitudinal axis of the second separation channel, said second electrode being connected to a source of potential, said source of electropotential being also connected to the isolated ground. The analyte concentrator can further comprise an analyte species detector positioned intermediate said first electrode and said second electrode.